WO1999020780A1 - Selection par gene marqueur positif ou negatif lors d'une recombinaison homologue - Google Patents

Selection par gene marqueur positif ou negatif lors d'une recombinaison homologue Download PDF

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Publication number
WO1999020780A1
WO1999020780A1 PCT/EP1998/006616 EP9806616W WO9920780A1 WO 1999020780 A1 WO1999020780 A1 WO 1999020780A1 EP 9806616 W EP9806616 W EP 9806616W WO 9920780 A1 WO9920780 A1 WO 9920780A1
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WIPO (PCT)
Prior art keywords
selection marker
negative selection
cell
gene
flanking
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PCT/EP1998/006616
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German (de)
English (en)
Inventor
Johannes Auer
Raimund Sprenger
Konrad Honold
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Roche Diagnostics Gmbh
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Priority to BRPI9813099-4A priority Critical patent/BR9813099B1/pt
Priority to US09/509,560 priority patent/US6284541B1/en
Priority to DE59810925T priority patent/DE59810925D1/de
Priority to AU12293/99A priority patent/AU730466B2/en
Priority to AT98955483T priority patent/ATE260982T1/de
Application filed by Roche Diagnostics Gmbh filed Critical Roche Diagnostics Gmbh
Priority to DK98955483T priority patent/DK1025253T4/da
Priority to KR1020007004264A priority patent/KR100566702B1/ko
Priority to EP98955483A priority patent/EP1025253B2/fr
Priority to CA2306229A priority patent/CA2306229C/fr
Priority to JP2000517100A priority patent/JP4417549B2/ja
Publication of WO1999020780A1 publication Critical patent/WO1999020780A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells

Definitions

  • the invention relates to a method for introducing a foreign DNA into the genome of a target cell by homologous recombination and DNA constructs suitable for homologous recombination.
  • a starting cell is transfected with a DNA construct that contains at least one, preferably two, DNA sequence sections that are homologous to regions of the genome that are to be trans-
  • the DNA construct can contain a heterologous expression control sequence if a gene that is normally silent in the transfected cell is to be activated.
  • the transfected cells are cultured under conditions where one
  • a second selection step is usually carried out. This is done by using a negative selection marker gene, such as the HSV thymidine kinase gene (HSV-TK), in the presence of which cells are present in the presence of a selection agent, e.g. Ganciclovir, to be destroyed. With homologous recombination, the cell loses
  • a negative selection marker gene such as the HSV thymidine kinase gene (HSV-TK)
  • HSV-TK HSV thymidine kinase gene
  • HSV thymidine kinase gene so that cells are resistant to ganciclovir.
  • Cells in whose genome the targeting vector was inserted by random, non-homologous integration do not lose the HSV-TK gene and are therefore sensitive to ganciclovir.
  • HSV-TK / GancicIovir preference is given to using cells which do not contain a functional thymidine kinase gene (for example CEM tk " from Ogden Bioservices Corp., Rockville MD, USA, Cat. No. 491).
  • polypeptides located on the cell surface as a positive transfection marker is known.
  • WO 95/06723 describes a method for labeling cells using a partially deleted cell surface receptor gene.
  • a negative selection marker gene is used according to the invention which codes for a polypeptide located on the cell surface.
  • the present invention thus relates to a method for introducing foreign DNA into a host cell by homologous recombination, the host cell being transfected with a recombinant vector comprising two flanking nucleotide sequences which are homologous to a target sequence in the genome of the host cell and within which a nucleotide sequence coding for a positive selection marker is located, and outside the flanking sequences there is a nucleotide sequence coding for a negative selection marker, the nucleotide sequences coding for the positive and negative selection markers in each case are operatively linked to an expression control sequence active in the host cell, at least one nucleotide sequence coding for a polypeptide located on the cell surface being used as the negative selection marker gene, so that after integration of the DNA construct by homologous recombination into the genome of the cell, the negative selection marker gene is not is expressed and after a random integration of the vector into the genome of the cell, the negative selection marker gene is expressed and its gene product is presented on the
  • a negative selection marker gene coding for a surface-localized polypeptide is used at a corresponding point in the vector for the homologous recombination.
  • a negative selection marker gene is used which codes for a polypeptide which is normally not found in the host cell.
  • the method according to the invention does not give rise to problems with toxicity or with background signals, as described in the case of TC selection.
  • Another advantage of the method according to the invention is that the number of transfected cells that have to be examined for the expression of the target gene is significantly reduced.
  • the host cell is preferably a eukaryotic cell, more preferably a mammalian cell, and most preferably a human cell.
  • a selection step according to the invention is carried out for the presence of the positive selection marker gene and a further selection step for the absence of the negative selection marker gene.
  • the selection step for the presence of the positive selection marker gene can be carried out in the usual way.
  • Any positive marker gene particularly suitable for eukaryotic cells, can be used as the positive selection marker gene, which leads to a selectable phenotype when expressed, for example antibiotic resistance, or auxotrophy.
  • Antibiotic resistance genes are preferably used, for example the neomycin, kanamycin, geneticin or hygromycin resistance gene.
  • a particularly preferred positive selection marker gene is the neomycin phosphotransferase gene.
  • the negative selection marker gene used for the method according to the invention codes for a gene product which is presented on the surface of the host cell, preferably for a membrane-bound polypeptide.
  • membrane-bound polypeptides are, for example, the LNGF, the CD24, the LDL or the trk receptor or a membrane-bound receptor fragment which contains the ligand-binding domain of the respective receptor.
  • Suitable receptor fragments in which the intracellular domain has been completely or partially deleted or modified in such a way that the receptor presented on the surface cannot bring about signal transduction are described in WO 95/06723.
  • a particularly preferred example of such a receptor fragment is a deletion mutant of the LNGF receptor (dLNGFR), which is a fragment of the human low-affinity receptor of the nerve growth factor whose intracellular and signal-transducing domain has been deleted (WO 95/06723).
  • dLNGFR LNGF receptor
  • a plasmid is used as the recombinant vector which comprises two flanking nucleic acid segments (HR1, HR2) and in between the positive selection marker gene containing the neomycin resistance gene (NeoR). Outside the two flanking homologous nucleotide sequences, a nucleotide sequence coding for dLNGFR is arranged on the plasmid.
  • the regions HR1, NeoR and HR2 are integrated into the genome.
  • the sequence coding for dLNGFR is not integrated into the genome.
  • the dLNGFR gene is retained in an expressible form.
  • the selection according to the invention for the absence of the negative selection marker gene in the transfected host cell preferably comprises the steps:
  • binding molecules which can enter into a specific and preferably high affinity binding with the negative selection marker are used as binding molecules. Preference is given to using binding molecules which have no interfering cross-reactivity with other surface components of the host cell.
  • binding molecules are antibodies, for example polyclonal or monoclonal antibodies, antibody fragments, etc., which are directed against the gene product of the negative selection marker gene. Suitable antibodies against dLNGFR are known for example from WO 95/06723.
  • a receptor is used as a negative selection marker, it is of course also possible to use a natural binding partner of the receptor, for example the receptor ligand, or an analogue thereof as the binding molecule.
  • An example of such a receptor ligand are NGF as the ligand of LNGFR.
  • a binding molecule can be used which is coupled to a solid phase, which coupling can be carried out by adsorption, covalent binding or via a high-affinity binding pair (eg streptavidin / - biotin).
  • the type of solid phase is generally not critical for the method according to the invention; preference is given to using solid phases which make it easy to separate the cells presenting the negative selection marker from unlabeled cells.
  • the solid phase can therefore be in the form of a chromatography column, for example, but particulate solid phases such as microbeads, in particular magnetic microbeads, which allow particularly simple separation, are particularly preferred.
  • the transfected cells can also be brought into contact with free binding molecules.
  • the free binding molecules preferably carry a labeling or / and a solid phase binding group.
  • Biotin biotin derivatives, e.g. Iminobiotin, aminobiotin or desthiobiotin,
  • Haptens e.g. Digoxigenin, fluorescein, enzymes, e.g. Peroxidase or alkaline phosphatase or dyes, e.g. Fluorescent dyes such as
  • the cell labeled with the binding molecule can be coupled to a solid phase which can react with the solid phase binding group of the binding molecule.
  • the cells expressing the negative selection marker can be identified by binding to an avidin- or streptavidin-coated solid phase and separated from unlabeled cells.
  • the cells expressing the negative selection marker can be identified by adding an enzyme substrate by an enzyme-catalyzed color reaction and, if necessary, separated from unlabeled cells 5. This identification can take place, for example, by applying the cells to a slide and subsequent microscopic analysis.
  • the cells expressing the negative selection marker can be identified by flow cytometric analysis and separated from unlabeled cells. This separation process is quick and easy and can be carried out in standard FACS devices, which allow the setting of fluorescence windows and cell sorting.
  • Another object of the present invention is a recombinant vector which is suitable for use as a transfection vector in the method according to the invention.
  • This vector includes:
  • nucleotide sequence coding for a negative selection marker under the control of an expression control sequence active in the cell which is located outside the flanking homologous nucleotide sequences and whose expression product is a polypeptide located on the cell surface.
  • the recombinant vector If the recombinant vector is to be used to activate a gene endogenously present in the host cell, it contains between the two flanking homologous nucleotide sequences and a heterologous expression control sequence that is active in the host cell.
  • This expression control sequence comprises a promoter and preferably further expression-improving sequences, for example an enhancer.
  • the promoter can be an adjustable or a consecutive promoter.
  • the promoter is preferably a strong viral promoter, for example an SV40 or a CMV promoter.
  • the CMV promoter / enhancer is particularly preferred.
  • the recombinant vector contains an amplification gene between the two flanking sequences.
  • suitable amplification genes are dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase etc.
  • a particularly preferred amplification gene is the dihydrofolate reductase gene, in particular a gene coding for a dihydrofolate reductase-arginine mutant which has a lower sensitivity for the selective agent type (Methot) than the wild type solvent (Methot) Simonsen et al., Proc. Natl. Acad. Sci. USA 80 (1 983), 2495).
  • the nucleotide sequence coding for the negative selection marker can - as explained above - preferably be selected from membrane-bound receptors or membrane-bound receptor fragments containing the ligand-binding domain of the respective receptor.
  • flanking nucleotide sequences homologous to a target sequence can be selected from any chromosomal regions of the genome of the cell to be transfected, which is preferably a eukaryotic cell, particularly preferably a mammalian cell and most preferably a human cell.
  • the flanking homologous nucleotide sequences will preferably originate from the genes for human factors, for example EPO, tPA, G-CSF, GM-CSF, TPO, interleukins, interferons, growth factors, insulin, insulin-like growth factors etc.
  • the flanking homologous nucleotide sequences can comprise the coding region of the target gene or a part thereof.
  • they can be selected so that, when homologous recombination occurs, they cause a mutation in the coding region of the mature target polypeptide with respect to the sequence present endogenously in the cell.
  • This mutation can include substitutions, deletions and insertions of individual amino acids or entire amino acid segments.
  • Yet another object of the present invention is the use of membrane-based surface receptors as negative selection markers in a homologous recombination process.
  • Figure 1 A schematic representation of the principle of homologous recombination using a negative selection by dLNGFR according to the invention
  • FIGS. 3a and b results of a FACS analysis of cells expressing and not expressing dLNGFR
  • Figure 4 the restriction map of plasmid p 1 87-dLNGFR
  • Figure 5 the result of a FACS analysis to distinguish dLNGFR negative and positive cells
  • the vector was dissolved in a concentration of 1 ⁇ g / ⁇ l bidistilled water.
  • the cells were transfected with the help of electroporation (BioRad, Genepulser TM) under previously determined optimal conditions (960 ⁇ F / 260 MV / 1 8-22 ⁇ S).
  • the adherently growing human fibrosarcoma line HT1 080 was used as a suitable cell line in a concentration of 1 0 7 cells / 0.8 ml. Before and after the transfection, the cells were kept on ice for about 10 minutes in order to reconstitute the cell membrane.
  • Transfected cells were sown in T-1 75 culture bottles and cultured at 37 ° C and 7% C0 2 in the incubator. After 24 h, selection pressure was applied by adding G41 8 (0.8 ⁇ g / ml).
  • the staining steps were carried out with 10 5 cells / batch on ice.
  • the mouse anti-dLNGFR antibody added as primary antibody was detected by adding a goat secondary antibody ( ⁇ -mlgG-FITC, 1:25, cold day).
  • a goat secondary antibody ⁇ -mlgG-FITC, 1:25, cold day.
  • Dead cells were detected by adding propidium iodide (10 ⁇ g / ml).
  • the analyzes were carried out on a FACS-Vantage (from Becton Dickinson) according to the manufacturer's instructions.
  • the specific fluorescence of the cells expressing dLNGFR was recorded in the FL-1 channel, the dead cells in the FL-3 channel.
  • the gene for dLNGFR (WO 95/06723, Boehringer Mannheim GmbH), which comprises 965 bp, was amplified using the PCR technique.
  • the primers used introduced interfaces for the enzymes EcoRI and Sall at both ends. After amplification, the PCR fragments were cut with both enzymes.
  • the vector pSV 1 which contains the early SV40 promoter and the SV40 polyA signal (Okayama and Berg, Mol. Cell. Biol. 3 (1 983), 280-289; Mulligan and Berg, Proc. Natl. Acad. Sei. USA 78 (1 981), 2072-2076) was also cut with EcoRI and Sall. The isolated vector has a size of 3490 bp. The dLNGFR fragment was ligated into the vector pSV1. The gene for dLNGFR is under the expression control of the SV40 early promoter and the SV40 poly signal. The entire expression cassette comprises 1 900 bp. The resulting vector pSV-DLNGR is shown in Fig. 2.
  • the dLNGFR expression cassette was isolated from pSV-DLNGFR with the restriction enzymes Notl and Pvull.
  • the targeting vector 'p 1 87' for the human EPO gene (described in EP 97 1 1 649.5 and EP 97 1 1 2 640.5 see Fig. 4b) was cut with Notl and EcoRV.
  • the 1 4551 bp vector fragment was isolated and ligated to the dLNGFR expression cassette (FIG. 4).
  • the resulting plasmid 'p 1 87-DLNGFR' was transferred to E. coli and propagated therein.
  • HT1 080 cells were transfected with p 1 87-DLNGFR and selected for stable integration, ie G41 8 was added to the medium 24 hours after the transfection.
  • the first FACS analysis was carried out after about 3 weeks of growth and after the formation of the first foci, the cells of which were pooled.
  • dLNGFR-negative cells here 1 4% of the population, can be distinguished from the dLNGFR-expressing cells by FACS analysis.
  • this cell population also contains cells that have too low a receptor density on their surface and are therefore not recognized by the detection system. In this way, however, the number of clones that must subsequently be tested for expression of the target gene can be significantly reduced (here 1 4 out of 1 00%).

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Abstract

L'invention concerne un procédé d'introduction d'un ADN étranger dans le génome d'une cellule cible par recombinaison homologue, ainsi que des constructions d'ADN pouvant être utilisées pour la recombinaison homologue.
PCT/EP1998/006616 1997-10-20 1998-10-20 Selection par gene marqueur positif ou negatif lors d'une recombinaison homologue WO1999020780A1 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
JP2000517100A JP4417549B2 (ja) 1997-10-20 1998-10-20 相同組換えにおける陽性−陰性選択
US09/509,560 US6284541B1 (en) 1997-10-20 1998-10-20 Positive-negative selection for homologous recombination
DE59810925T DE59810925D1 (de) 1997-10-20 1998-10-20 Positiv-negativ-selektion bei der homologen rekombination
AU12293/99A AU730466B2 (en) 1997-10-20 1998-10-20 Positive-negative selection for homologous recombination
AT98955483T ATE260982T1 (de) 1997-10-20 1998-10-20 Positiv-negativ-selektion bei der homologen rekombination
BRPI9813099-4A BR9813099B1 (pt) 1997-10-20 1998-10-20 processo para a introduÇço de dna exàgeno em uma cÉlula hospedeira mediante recombinaÇço homàloga, vetor recombinante, bem como emprego do referido vetor e de receptores superficiais em posiÇço de membrana.
DK98955483T DK1025253T4 (da) 1997-10-20 1998-10-20 Positiv-negativ-selektion ved den homologe rekombination
KR1020007004264A KR100566702B1 (ko) 1997-10-20 1998-10-20 상동 재조합에 있어서 양성-음성 선별법
EP98955483A EP1025253B2 (fr) 1997-10-20 1998-10-20 Selection par gene marqueur positif ou negatif lors d'une recombinaison homologue
CA2306229A CA2306229C (fr) 1997-10-20 1998-10-20 Selection par gene marqueur positif ou negatif lors d'une recombinaison homologue

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP97118175.5 1997-10-20
EP97118175 1997-10-20

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WO1999020780A1 true WO1999020780A1 (fr) 1999-04-29

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US (1) US6284541B1 (fr)
EP (1) EP1025253B2 (fr)
JP (1) JP4417549B2 (fr)
KR (1) KR100566702B1 (fr)
CN (1) CN1146666C (fr)
AR (1) AR017368A1 (fr)
AT (1) ATE260982T1 (fr)
AU (1) AU730466B2 (fr)
BR (1) BR9813099B1 (fr)
CA (1) CA2306229C (fr)
DE (1) DE59810925D1 (fr)
DK (1) DK1025253T4 (fr)
ES (1) ES2213925T5 (fr)
PT (1) PT1025253E (fr)
TR (1) TR200001046T2 (fr)
WO (1) WO1999020780A1 (fr)
ZA (1) ZA989497B (fr)

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WO2002022834A2 (fr) * 2000-09-15 2002-03-21 Deltagen, Inc. Procedes permettant de produire des cellules et des animaux comprenant la modification d'un gene cible et compositions associees
WO2002055687A3 (fr) * 2000-12-13 2003-05-22 Deltagen, Inc. Souris transgeniques presentant des disruptions geniques cibles
EP1373473A1 (fr) * 2001-12-04 2004-01-02 Genome Biosciences, LLC Procedes et vecteurs de ciblage genetique
FR2860517A1 (fr) * 2003-10-03 2005-04-08 Evologic Sa Procedes et outils pour la modification chromosomique de bacteries
EP2045327A2 (fr) 2005-03-08 2009-04-08 BASF Plant Science GmbH Expression à amélioration de séquences d'intron
US8771960B2 (en) 2001-10-01 2014-07-08 Deutsches Krebsforschungszentrum Method for producing protein libraries and for selecting proteins from said libraries

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WO2003027261A2 (fr) * 2001-09-27 2003-04-03 Functional Genetics, Inc. Procedes et compositions de ciblage de gene par recombinaison homologue
WO2003093426A2 (fr) * 2002-05-02 2003-11-13 University Of North Carolina At Chapel Hill Mutagenese in vitro, phenotypage et cartographie genetique
PE20081216A1 (es) 2006-09-01 2008-09-04 Therapeutic Human Polyclonals Inc Expresion mejorada de la inmunoglobulina humana o humanizada en animales transgenicos no humanos
EP2147105B1 (fr) 2007-05-02 2013-04-24 Merial Limited Plasmides à adn à expression et stabilité améliorées
US20110136236A1 (en) * 2008-04-21 2011-06-09 Unitargeting Research As Genetically modified eukaryotic cells
CN102618453A (zh) * 2011-10-31 2012-08-01 四川农业大学 猪传染性胸膜肺炎放线杆菌Apx I C基因缺失突变株、构建方法、疫苗及应用
US9303272B2 (en) 2011-12-13 2016-04-05 Public University Corporation Yokohama City University Gene targeting vector, and method for using same
DE102014116334A1 (de) * 2014-11-10 2016-05-12 Eberhard Karls Universität Tübingen Medizinische Fakultät Herstellung von rekombinanten Expressionsvektoren

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WO2002022834A3 (fr) * 2000-09-15 2003-01-30 Deltagen Inc Procedes permettant de produire des cellules et des animaux comprenant la modification d'un gene cible et compositions associees
US6929909B2 (en) 2000-09-15 2005-08-16 Deltagen, Inc. Gene targeting vectors comprising conditional positive selection markers
WO2002022834A2 (fr) * 2000-09-15 2002-03-21 Deltagen, Inc. Procedes permettant de produire des cellules et des animaux comprenant la modification d'un gene cible et compositions associees
WO2002055687A3 (fr) * 2000-12-13 2003-05-22 Deltagen, Inc. Souris transgeniques presentant des disruptions geniques cibles
US8771960B2 (en) 2001-10-01 2014-07-08 Deutsches Krebsforschungszentrum Method for producing protein libraries and for selecting proteins from said libraries
EP1298207B2 (fr) † 2001-10-01 2015-12-30 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Méthodes pour construire des banques de protéines et pour isoler des protéines de ces banques
EP1373473A1 (fr) * 2001-12-04 2004-01-02 Genome Biosciences, LLC Procedes et vecteurs de ciblage genetique
EP1373473A4 (fr) * 2001-12-04 2005-02-16 Genome Biosciences Llc Procedes et vecteurs de ciblage genetique
FR2860517A1 (fr) * 2003-10-03 2005-04-08 Evologic Sa Procedes et outils pour la modification chromosomique de bacteries
WO2005033322A1 (fr) * 2003-10-03 2005-04-14 Consortium National De Recherche En Genomique Procedes et outils pour la modification chromosomique de bacteries
EP2045327A2 (fr) 2005-03-08 2009-04-08 BASF Plant Science GmbH Expression à amélioration de séquences d'intron
EP2166100A2 (fr) 2005-03-08 2010-03-24 BASF Plant Science GmbH Séquences introniques facilitant l'expression
EP2166101A2 (fr) 2005-03-08 2010-03-24 BASF Plant Science GmbH Séquences introniques facilitant l'expression
EP2169058A2 (fr) 2005-03-08 2010-03-31 BASF Plant Science GmbH Expression à amélioration de séquences d'intron
EP2166099A2 (fr) 2005-03-08 2010-03-24 BASF Plant Science GmbH Expression à amélioration de séquences d'intron
EP2166102A2 (fr) 2005-03-08 2010-03-24 BASF Plant Science GmbH Expression à amélioration de séquences d'intron

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ES2213925T3 (es) 2004-09-01
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DK1025253T4 (da) 2009-08-31
AU730466B2 (en) 2001-03-08
ATE260982T1 (de) 2004-03-15
AU1229399A (en) 1999-05-10
DE59810925D1 (de) 2004-04-08
JP4417549B2 (ja) 2010-02-17
CN1146666C (zh) 2004-04-21
ZA989497B (en) 2000-04-19
KR100566702B1 (ko) 2006-04-03
EP1025253B1 (fr) 2004-03-03
PT1025253E (pt) 2004-07-30
ES2213925T5 (es) 2009-11-11
US6284541B1 (en) 2001-09-04
EP1025253A1 (fr) 2000-08-09
CN1276836A (zh) 2000-12-13
DK1025253T3 (da) 2004-07-12
BR9813099B1 (pt) 2010-02-09
CA2306229A1 (fr) 1999-04-29
BR9813099A (pt) 2000-08-22
AR017368A1 (es) 2001-09-05
KR20010031277A (ko) 2001-04-16
TR200001046T2 (tr) 2001-07-23
EP1025253B2 (fr) 2009-07-29

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